Ribisi S et al. (2000), Ras-mediated FGF signaling is required for the ...

XB-ART-9988

Dev Biol 2000 Nov 01;2271:183-96. doi: 10.1006/dbio.2000.9889.

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Ras-mediated FGF signaling is required for the formation of posterior but not anterior neural tissue in Xenopus laevis.

Ribisi S , Mariani FV , Aamar E , Lamb TM , Frank D , Harland RM .

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Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, we have employed the dominant negative Ras mutant, N17Ras, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a dusp6 eef1a2 egr2 en2 fgf2 foxa1 foxd1 hoxb9 mapk1 meis3 myod1 ncam1 neurog1 nog nrp1 otx2 sox2

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	FIG. 1. The ability of FGF to induce posterior neural tissue and to posteriorize existing neural tissue is mediated through Ras, but Ras-mediated FGF signaling is not required for anterior neural induction. (A) RT-PCR of animal caps treated with soluble noggin, bFGF, or both in the presence or absence of N17Ras. Stage 25 whole-embryo positive control (lane 1) and RT2 negative control (lane 2) were both derived from total RNA isolated from a normal embryo. Animal caps without N17Ras (lanes 3–6) and animal caps injected with 1 ng of N17Ras mRNA (lanes 7–10) were subjected to RT-PCR for various region-specific A-P neural markers. (B) RT-PCR of animal caps injected with 100 pg of mRNA encoding Xenopus noggin (lane 4) or 100 pg noggin in combination with 1 ng of inactive FGF receptor mRNA (HAVf, lane 5), 1 ng dominant negative FGF receptor (XFD, lane 6), 1 ng wild-type c-Ras (lane 7), or 1 ng dominant negative Ras (N17Ras, lane 8). (C) RT-PCR of animal caps injected with 100 pg of mRNA encoding Xenopus neurogenin (ngn) in combination with 1 ng XFD or 1 ng N17Ras mRNA. Uninjected animal caps (lane 3), neurogenin alone (lane 4), neurogenin and XFD (lane 5), and neurogenin and N17Ras (lane 6).
	FIG. 2. Noggin does not require Ras-mediated FGF signaling to induce anterior neural tissue. Whole-mount in situ hybridizations of animal caps explanted from uninjected embryos (A, E, I) and embryos injected with 100 pg Xenopus noggin mRNA alone (B, F, J, M), 100 pg noggin in combination with 1 ng XFD mRNA (C, G, K, N), or 100 pg noggin in combination with 1 ng N17Ras mRNA (D, H, L, O). Markers examined include (A–D) MyoD, (E–H) Nrp1, and (I–L) Otx2. A, E, and I are photographed at half the magnification relative to the others.
	FIG. 3. N17Ras blocks the activation of the embryonic wounding response in animal caps. A commercially available antibody directed against the bisphosphorylated forms of MAPK (ERK1 and ERK2, see Materials and Methods) reveals the activation of the embryonic wounding response in explanted animal caps. The phosphorylation of MAPK is visualized in animal caps derived from uninjected control embryos at (A) 1, (B) 5, (C) 10, and (D) 20 min after explantation. MAPK phosphorylation is maximal at 10 min and is visible in the cells at the edges of the tissue. The phosphorylation of MAPK is largely reversed by 20 min after explantation. The activation of the wounding response is not prevented by 1 ng of c-Ras mRNA (E), but is blocked by 1 ng of N17Ras mRNA (F). Animal caps derived from embryos injected with 100 pg of Xenopus noggin mRNA alone (G), in combination with 1 ng of XFD mRNA (H), or in combination with 1 ng of N17Ras mRNA (I). The phosphorylation of MAPK in response to the removal of the animal caps is visible in the presence of noggin alone or noggin in combination with XFD but not when noggin is co-injected with N17Ras. The animal caps pictured in E–I were fixed 10 min after explantation.
	FIG. 4. Induction of posterior neural tissue by XBF2 and XMeis3 requires Ras-mediated FGF signaling. Markers used include Otx2, NCAM, Engrailed 2 (En2), hoxB9, HoxD, Krox20, muscle actin (MA), and EF1a as a control for loading. (A) RT-PCR of animal caps injected with XBF2 in combination with either XFD or N17Ras. RT-PCR was done on RNA isolated from 10 animal caps per condition. Stage 25 whole-embryo positive control (lane 1), RT2 negative control (lane 2), 100 pg XBF2 mRNA (lane 3), 100 pg XBF2 mRNA in combination with 1 ng XFD mRNA (lane 4), or 100 pg XBF2 mRNA in combination with 1 ng N17Ras mRNA (lane 5). (B) RT-PCR of animal caps injected with mRNA encoding XMeis3 in combination with N17Ras. RNA was isolated from 18 animal caps per condition. Stage 20 whole-embryo positive control (lane 1), uninjected animal caps (lane 2), 1 ng XMeis3 alone (lane 3), 1 ng N17Ras alone (lane 4), 1 ng XMeis3 in combination with 1 ng N17Ras (lane 5), and RT2 negative control (lane 6). (C) RT-PCR of animal caps injected with mRNA encoding XMeis3 in combination with MAP kinase phosphatase (MKP). RNA was isolated from 18 animal caps per condition. Stage 20 whole-embryo positive control (lane 1), uninjected animal caps (lane 2), 1 ng XMeis3 (lane 3), 4 ng MKP (lane 4), 1 ng XMeis3 in combination with 4 ng MKP (lane 5), and RT2 negative control (lane 6).
	FIG. 5. Disruption of Ras-mediated FGF signaling affects the expression of Sox2 in the posterior neural plate. b-Galactosidase activity stain (red) in the neural plates of embryos stained for Sox2 expression (blue) by in situ hybridization. (A) Uninjected control embryo in which the normal expression domain of Sox2 is visible. Embryos injected with lacZ mRNA in addition to XFD (B, C), c-Ras (D), or N17Ras (E, F). If XFD (B) or N17Ras (E) injected mRNAs are expressed in the anterior portion of the neural plate then the expression of the general neural marker Sox2 is unaffected compared to the control of c-Ras (D). However, if XFD (C) or N17Ras (F) mRNAs are expressed in the posterior portion of the neural plate, disruptions of Sox2 staining result. All embryos are oriented with the anterior neural plate facing upward. Arrows indicate loss of Sox2 expression in the posterior neural plate.
	FIG. 6. Grafted neural plates expressing XFD or N17Ras exhibit disruption of posterior neural patterning. (A) A schematic diagram illustrating the neural plate graft technique. (B) A photo of a graft-recipient embryo shortly after the grafted tissue has healed in place. The darkly pigmented ectodermal graft is visible on one side of the host embryo, near the dorsal blastopore lip which is oriented up. (C–E) 1 ng of RNA encoding nuclear b-galactosidase was injected into the animal pole of one-cell embryos. b-Galactosidase activity (red) was used to indicate the distribution of injected RNA in whole donor embryos (C), embryos after grafts have been removed (D), and isolated grafts (E). b-Galactosidase activity is visible throughout the animal pole of the donor embryos and is clearly visible throughout the entire graft. (F, H, J) The location of grafted tissue is revealed by the fluorescence of the Texas red dextran. (G, I, K) Sox2 expression in the same embryos. (F, G) c-Ras-expressing graft in which the expression of Sox2 is normal. (H, I) XFD-expressing graft and (J, K) N17Ras-expressing graft in which the expression of Sox2 is disrupted in the posterior but not anterior neural plate. (L) Cell expressing XFD in the spinal cord.